A molecular mechanism for probabilistic bet hedging and its role in viral latency Sonali Chaturvedia,1, Jonathan Kleina,1,2, Noam Vardia,1, Cynthia Bolovan-Frittsa,1, Marie Wolfa, Kelvin Dua, Luwanika Mlerab, Meredith Calverta, Nathaniel J. Moormanc, Felicia Goodrumb, Bo Huangd,e, and Leor S. Weinbergera,d,e,3 aGladstone Institute for Virology and Immunology, GladstonejUniversity of California, San Francisco Center for Cell Circuitry, San Francisco, CA 94158; bDepartment of Cell & Molecular Medicine, University of Arizona, Tucson, AZ 85721; cDepartment of Microbiology & Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; dDepartment of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158; and eDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94158 Edited by Thomas Shenk, Princeton University, Princeton, NJ, and approved June 3, 2020 (received for review August 26, 2019) Probabilistic bet hedging, a strategy to maximize fitness in a silenced state or initiate lytic replication, expression from CMV’s unpredictable environments by matching phenotypic variability to Major Immediate-Early Promoter (MIEP) is essential. During environmental variability, is theorized to account for the evolution silencing, the MIEP is largely quiescent, but during lytic replica- of various fate-specification decisions, including viral latency. How- tion the MIEP drives expression of crucial viral genes including ever, the molecular mechanisms underlying bet hedging remain un- the 86-kDa Immediate-Early 2 (IE2) protein, a master regulator clear. Here, we report that large variability in protein abundance of lytic viral expression. Initiation of lytic replication requires that within individual herpesvirus virion particles enables probabilistic the MIEP be efficiently transactivated by proteins carried within bet hedging between viral replication and latency. Superresolution the viral tegument (18), a proteinaceous region between the viral imaging of individual virions of the human herpesvirus cytomegalo- capsid and envelope. The pp150 (UL32) and pp71 (UL82) tegu- virus (CMV) showed that virion-to-virion levels of pp71 tegument ment proteins appear to be the chief tegument regulators of MIEP protein—the major viral transactivator protein—exhibit extreme var- expression (19–21), with pp71 transactivating MIEP activity and iability. This super-Poissonian tegument variability promoted alternate pp150 repressing MIEP activity. replicative strategies: high virion pp71 levels enhance viral replicative Building off previous quantitative analyses of tegument vari- fitness but, strikingly, impede silencing, whereas low virion pp71 levels ability in other herpesviruses (22–24), we quantified CMV single- reduce fitness but promote silencing. Overall, the results indicate that virion variability in pp71 and pp150 levels using superresolution stochastic tegument packaging provides a mechanism enabling fluorescence microscopy, and then examined how variability in probabilistic bet hedging between viral replication and latency. particle-associated protein abundance affects CMV replicative strategies. We find that virion-associated pp71 levels are signif- stochastic variability | fate selection | herpesvirus | latency | tegument icantly more variant than pp150 levels or than expected by Poisson statistics. When virions with increased pp71 levels were iverse biological systems share a common challenge to preserve generated, the population exhibited enhanced infectiousness and Dreproductive fitness in unpredictable, changing environments. replicative fitness. This enhanced fitness conferred by higher pp71 Faced with environmental variability, some organisms probabi- raised the question of why selective pressures had not forced the listically generate a range of phenotypes to “hedge their bets” (1–7), in much the same way that financial houses diversify their Significance assets to minimize risk against economic crashes. First proposed — over 50 years ago (1) for desert annuals where reproductive Probabilistic bet hedging is a generalized diversification strat- — success is subject to unpredictable weather patterns bet hedging egy to maximize fitness in unpredictable environments and has theory noted that temporal variation in fitness could be minimized been proposed as an evolutionary basis for herpesvirus la- if husk thickness between seeds varied such that random chance tency. However, the molecular mechanisms enabling probabi- biased the population’s germination potential. In this way, some listic bet hedging have remained elusive. Here, we find that the seeds randomly enter dormancy irrespective of the environment, human herpesvirus cytomegalovirus—a major cause of birth and a long-lived, desiccation-resistant subpopulation is always defects and transplant failures—utilizes stochastic variability in formed to avoid extinction during unforeseen droughts, but at the the abundance of a protein packaged into individual viral necessary expense of lowering germinative fitness. In the decades particles to enable probabilistic bet hedging between alternate since, bet hedging has been studied in persistence phenotypes in viral states. bacteria (2, 5, 7, 8), yeasts (9, 10), and viruses (11–13). In her- pesviruses, bet hedging theory has been proposed as a theoretical Author contributions: S.C., J.K., C.B.-F., and L.S.W. designed research; S.C., J.K., N.V., C.B.-F., basis for the evolution of viral latency (11) and associated viral M.W., and K.D. performed research; L.M., M.C., N.J.M., and F.G. contributed new re- agents/analytic tools; S.C., J.K., N.V., C.B.-F., M.W., B.H., and L.S.W. analyzed data; and gene silencing. However, the molecular mechanisms that allow S.C., J.K., N.V., and L.S.W. wrote the paper. biological systems, such as viruses, to probabilistically generate The authors declare no competing interest. the needed variability have remained unclear and an area of This article is a PNAS Direct Submission. active study. This open access article is distributed under Creative Commons Attribution-NonCommercial- In the herpesviridae family, persistence within the host is NoDerivatives License 4.0 (CC BY-NC-ND). mediated by establishment of a reversible latent state, wherein 1S.C., J.K., N.V., and C.B.-F. contributed equally to this work. viral gene expression is largely silenced (14, 15). In human cy- 2 — Present address: Medical Scientist Training Program, Yale University, New Haven, tomegalovirus (CMV) one of nine human herpesviruses and a CT 06519. — leading cause of birth defects and transplant failure lytic rep- 3To whom correspondence may be addressed. Email: leor.weinberger@gladstone. lication occurs in a variety of cell types, while latency and requisite ucsf.edu. silencing are established in myeloid-progenitor cells (14, 16, 17). This article contains supporting information online at https://www.pnas.org/lookup/suppl/ The silent state is thought to enable evasion of host–immune re- doi:10.1073/pnas.1914430117/-/DCSupplemental. sponses and confer a selective advantage (11). To reactivate from First published July 6, 2020. 17240–17248 | PNAS | July 21, 2020 | vol. 117 | no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1914430117 Downloaded by guest on September 26, 2021 pp71 distribution to a higher mean and lower variance, indicating analysis, and a minimum of 2,000 intact, singlet particles were that a putative counterbalancing selection pressure may exist to quantified for each fusion virus. To control for contributions from maintain the broad pp71 distribution. Indeed, we found that low instrument and photon shot noise, a “molecular ruler” (27), con- pp71 levels promoted MIEP silencing, whereas high pp71 levels taining precisely 900 florescent-fused HSV-1 VP26 proteins per impeded silencing in undifferentiated cells. We propose a con- viral particle (24), was imaged in parallel and used for comparison. ceptual model for how heterogeneity in the levels of virion- Quantitative image analysis showed that the per-virion levels packaged tegument transactivators may enable probabilistic bet of the pp150 tegument protein, which is capsid-associated, fell hedging between replication and silencing in herpesviruses. within a relatively confined range, exhibiting two- to threefold particle-to-particle variation in intensity (Fig. 1B). This variation Results and Discussion in pp150 was similar to the virion-to-virion variation in HSV-1 The HCMV Major Tegument Transactivator Protein pp71 Exhibits Super- VP26 capsid levels (Fig. 1B), which are considered invariant Poissonian Variability in Virion-to-Virion Abundance. To measure the (24). However, in striking contrast, pp71 tegument levels imaged variation in pp71 and pp150 abundance in individual virion parti- under identical conditions and parameters varied >10-fold be- cles, we utilized a superresolution imaging method (25) that en- tween the dimmest and brightest virion particles (Fig. 1B), with abled fluorescent imaging of pp71 or pp150 tegument proteins that the normalized virion-to-virion variance (σ2/μ, a standardized were genetically tagged with yellow fluorescent protein (YFP) measure of the histogram width referred to as the “Fano factor”) (Fig. 1A). To obtain the viral samples for imaging, recombinant being fivefold greater for pp71 than pp150 (Fig. 1C). Importantly, virus was packaged in culture and viral preparations were gradient an orthogonal confocal imaging method showed close agreement